Major losses of crop yields and quality can result from infection of crops by plant disease pathogens including viruses, bacteria, and fungi. The tobacco mosaic virus (TMV) infects plants of commercial importance including tobacco and related plants such as tomato and pepper. While not lethal, TMV affects the growth and productivity of these plants. The virus pathogen spreads throughout the plant in two stages. First, virus infection occurs at the site where the virus is introduced into the cells of the host plant. Second, virus replication occurs wherein the virus multiplies within the cells of the plant.
Plants have numerous mechanisms which provide natural resistance to attack by pathogens. These include preformed structural and chemical barriers and active resistance mechanisms. Plant disease resistance to numerous pathogens is controlled by single complementary genes in the plant and the pathogen. The genes of the plant are termed resistance genes, and those of the pathogen are termed avirulence genes. Plants bearing a resistance gene are effectively protected from disease caused by a pathogen bearing the corresponding avirulence gene.
The dominant N locus of tobacco confers resistance to TMV and mediates a localized hypersensitive response (HR) at the site of viral infection and the induction of the systemic acquired resistance (SAR) response in cells neighboring the infection site and throughout the plant. Tobacco plants heterozygous or homozygous for the N locus are resistant to disease caused by TMV. The HR is a complex, active resistance response that is induced in the plant in response to pathogen attack after preformed resistance mechanisms fail (Keen et al., Biotechnology in Plant Disease Control, Wiley-Liss, Inc., pages 65-88 (1993)). HR is characterized by cell death (necrosis) at the site of pathogen ingress. Although necrosis per se may not be responsible for resistance to an invading pathogen, the concomitant syntheses of antimicrobial compounds, pathogenesis-related proteins that characterize the SAR response, and establishment of structural barriers are thought to play a central role in halting pathogen spread. Plant-pathogen interactions in which the outcome is resistance are termed incompatible, whereas those resulting in disease are compatible.
Studies have been carried out on the mechanisms by which plants carrying disease resistance genes discern the presence of an invading pathogen and invoke the HR and SAR. In many instances, the HR is governed by gene-for-gene interactions between incompatible plant and pathogen combinations. The gene model as proposed by Flor (Journal of Agricultural Research 74:241-262 (1947)) predicts that disease resistance and pathogen avirulence (the production of an elicitor) are dominant traits. Therefore, resistance will occur only in cases where the plant possesses the specific resistance gene (R gene) and the pathogen possesses the corresponding avirulence gene (Avr gene). Several Avr genes have been cloned from bacteria, fungi, and viruses [see Gabriel and Rolfe, Annual Rev. Phytopathology 28:365-391 (1990) and Keen, Annual Rev. Genet. 24:447-463 (1990)], and in some instances the nature of the elicitor molecule has been defined (see Keen, Plant Molecular Biology 19:109-122 (1992)). The fungal resistance gene, HM1, of maize (Johal and Briggs, Science 258:985-987 (1992)) and a bacterial resistance gene, Pro, of tomato (G. Martin et al., Science 262:1432-1436 (1993)) have been reported. No natural plant virus resistance gene has been isolated or cloned heretofore.
The simple genetic relationship between R genes and their corresponding Avr genes has led to speculation on the mode of action of R gene products. One model predicts that R genes lie in signaling pathways capable of recognizing pathogens and initiating subsequent signal transduction cascades leading to resistance (Lamb, Cell 76:419-422 (1994)). The second model predicts that R gene products are transmembrane ion channels that mediate cell death independent of other events in the cell. The recent cloning of Pto from tomato, conferring resistance to the bacterial pathogen Pseudomonas syringae pathovar tomato (Martin et al., Science 262:1432-1436 (1993)) suggests that at least the first model may be operating in plant cells. Sequence analysis of Pto indicates that it encodes a serine/threonine kinase. It is theorized that this serine/threonine kinase interacts directly or indirectly with the elicitor molecule and then phosphorylates a subsequent modulator of the resistance response, thereby initiating a signal transduction cascade.
Similarities have been noted between the hypersensitive resistance reponses of plants and the "innate" immune responses of animals. The unifying theme is the rapid production of reactive oxygen species (ROS), known as the oxidative burst. Examples of ROS are the superoxide anion (O.sub.2) and hydrogen peroxide (H.sub.2 O.sub.2). These molecules may have direct antimicrobial effects and other protective effects such as the crosslinking of structural proteins in the plant cell wall. Importantly, ROS can activate expression of defense-related genes in animals and plants (Schreck and Bauerle, Trends in Cell Biology 1:39-42 (1991), Chen et al., Science 262:1883-1886 (1993)). In mammals, ROS are strongly implicated as second messengers for cytokines such as tumor necrosis factor (TNF) and Interleukin-1(Il-1) in a pathway where the transcription factor NF-kB regulates the expression of immunoglobulins, interleukins and other proteins. A Drosophila transcription factor (Dif) homologous to NF-kB also activates transcription of antibacterial proteins including cecropins, attacins, defensins, and lysozymes (Levine and Hultmark, Trends in Genetics 9:178-183 (1993)). The parallel in plants is the induction of Pathogenesis Related Proteins and synthesis of antimicrobial compounds such as phytoalexins, which can be induced by exogenous application of H.sub.2 O.sub.2.
An important model system for the study of plant resistance responses has been that of the resistance gene N. The N locus is composed of a single dominant gene which mediates induction of a necrotic-type response and the SAR in response to infection by TMV (Holmes, Phytopathology 28:553-561 (1938)). It was originally identified in Nicotiana glutinosa, and has been introgressed into N. tabacum. The N gene mediates a hypersensitive response that is characterized by the formation of local lesions to which tobacco mosaic virus is localized. This is shown in FIG. 1A. Tobacco cultivars without the N gene allow tobacco mosaic virus to spread systemically and develop "mosaic" symptoms characterized by intermittant areas of light and dark green leaf tissue (FIG. 1B).
Recombinant DNA technology offers potential for obtaining plants transformed with a pathogen resistance gene to impart resistance. This approach has been impeded by lack of cloned natural plant resistance genes and by lack of knowledge of the mechanistic basis of resistance. Until recently, however, cloned resistance genes have been unavailable due to the lack of techniques in plants to isolate genes for which no information regarding the nature of the gene or its product is available. Two techniques have recently been developed for plants and do not depend on knowledge of gene or biochemical knowledge of protein have permitted isolation of genes these techniques are positional cloning and transposon tagging (Baker, Schell, Fedoroff, Proceedings of National Academy of Science, USA 83:4844-4848 (1986)).